The recognition of structured elements by a conserved groove distant from domains associated with catalysis is an essential determinant of RNase E

Abstract RNase E is an endoribonuclease found in many bacteria, including important human pathogens. Within Escherichia coli, it has been shown to have a major role in both the maturation of all classes of RNA involved in translation and the initiation of mRNA degradation. Thus, knowledge of the major determinants of RNase E cleavage is central to our understanding and manipulation of bacterial gene expression. We show here that the binding of RNase E to structured RNA elements is crucial for the processing of tRNA, can activate catalysis and may be important in mRNA degradation. The recognition of structured elements by RNase E is mediated by a recently discovered groove that is distant from the domains associated with catalysis. The functioning of this groove is shown here to be essential for E. coli cell viability and may represent a key point of evolutionary divergence from the paralogous RNase G family, which we show lack amino acid residues conserved within the RNA-binding groove of members of the RNase E family. Overall, this work provides new insights into the recognition and cleavage of RNA by RNase E and provides further understanding of the basis of RNase E essentiality in E. coli.


INTRODUCTION
The translation machinery is amongst the most abundant cellular components in all domains of life (1)(2)(3). In rapidly growing Escherichia coli ∼250 000-400 000 tRNAs deliver charged amino acids to 30 000-60 000 ribosomes in the translation of a dynamic pool of mRNA transcripts that at any moment number 1500-3000 (4,5). Under conditions of rapid growth, despite the RNA components of the translational machinery being relatively stable (6), the need to populate both daughter cells at each division dictates that the production of RNA components of the translation machinery is one of the major activities associated with cell growth. In E. coli, the tRNA genes are transcribed as either polycistronic or monocistronic transcripts, which undergo extensive processing on both the 5 and 3 side of the tRNAs to generate the mature forms that can be aminoacylated (7).
RNase E, an endoribonuclease that is found in many bacteria including important pathogens of humans and in some plant plastids (8), plays an important, if not essential, role in E. coli tRNA processing by separating the individual pre-tRNAs from polycistronic transcripts (9)(10)(11)(12). Sites at which RNase E cleaves tRNA precursor have been mapped in vivo (10,11), many with nucleotide resolution (10,13), and preparations of RNase E have been shown to be capable of cleaving at such sites in vitro (13,14). This, along with tRNAs being structurally stable and well defined (15,16), makes these precursors suitable substrates for dissecting the molecular basis of the recognition and cleavage of RNA by RNase E (see Supplementary Figure S1).
RNase E also has a major role in the turnover of the greater part of the mRNA pool in E. coli (8,13,17). Much of the earlier work on RNase E focused on the ability of 5 -untranslated regions (5 -UTRs) to affect the overall rate of decay of mRNA transcripts (18). This led to the discovery that RNase E can interact with 5monophosphorylated ends (19,20), which can be converted from 5 -triphosphorylated ends (21) by routes that appear to be absolutely dependent on RppH, an RNA 5 pyrophosphohydrolase (22,23). Whilst it has been established that the generation of 5 -monophosphorylated ends and their accessibility to RNase E can be determinants of RNA stability for some mRNAs, the rate of decay of many, if not most transcripts in cells lacking RppH is unaffected (22). This is further supported by the fact that RppH itself is nonessential in E. coli (22,24).
It has also been shown that RNase E can cleave many substrates rapidly independent of their 5 -phosphorylation status (13,14,25), and sites of RNase E cleavage have been mapped at distance from 5 -UTRs, e.g. within the 3 -UTRs of cspA and rpsO mRNA (26,27). The removal of Rho-independent transcription terminators greatly enhances degradation by 3 to 5 exonucleases (28). In the absence of translation, mRNAs are increasingly susceptible to RNase E cleavage internal to the protein-coding segment (29)(30)(31)(32). Contrary to previous thinking (32), sites of efficient RNase E cleavage independent of access to a 5 -monophosphorylated end appear to be common rather than special cases.
The catalytic activity of E. coli RNase E is a function of its N-terminal half (NTH) (33), which forms a tetramer via the dimerization of dimeric units (34,35). Each dimeric unit contain two symmetrical active sites, each located within a DNase I-like domain, which along with an S1 domain can form a channel that binds single-stranded RNA. Located adjacent to each channel is a pocket called the 5sensor that can bind a 5 -monophosphorylated end (34). The recognition of 5 -monophosphorylated ends by the 5sensor can contribute to the overall stability of an interaction between RNase E and a substrate (36,37). The absence of a 5 -monophosphorylated end does not appear to present an intrinsic barrier to RNase E cleavage provided overall binding is supported by other interactions. In work leading to the present study, access to adjacent but not contiguous single-stranded regions has been shown to be a requirement for at least some RNase E cleavages within the context of tRNA maturation (13,14). Here we present evidence that a recently discovered RNA-binding groove (38) distant from the domains of RNase E associated with catalysis (34,39) also has a critical role in the processing of tRNA, is essential for the viability of E. coli and may represent a key point of evolutionary divergence from the paralogous RNase G family (40,41). The wider implications of our findings beyond the processing of tRNA precursors are discussed.

Synthesis of RNA transcripts
Transcripts were synthesized in vitro using T7 RNA polymerase and polymerase chain reaction (PCR)-generated templates and purified, as described previously (13,14). The concentration and integrity of RNA samples were determined using a NanoPhotometer ® P-300 (Geneflow) and polyacrylamide gel electrophoresis (PAGE), respectively. The sequences of the primers used to generate cDNA templates are provided in Supplementary Table S1.

Site-directed mutagenesis
Site-directed mutagenesis was performed using a QuikChange Multi Site-Directed Mutagenesis Kit according to the manufacturer's instructions (Agilent). Primers for mutagenesis were designed using the online QuikChange Primer Design tool (https://www.agilent.com/store/primerDesignProgram.jsp) and are provided in Supplementary Table S2. Transformations were performed with 2 l of mutagenic reactions and 50 l of chemically competent XL10-Gold cells (Agilent). Successful mutagenesis was confirmed by Sanger sequencing (GeneWiz).

Purification of NTH-RNase E and discontinuous cleavage assays
Recombinant polypeptides corresponding to the NTH of E. coli RNase E (residues 1-529) were purified as described previously (34). All were tagged with oligohistidine at their N-terminus. Polypeptides with wild-type sequence, T170V substitution and D346N substitution have been described previously (35). New to this study was a polypeptide with eight substitutions (R3Q, Q22N, H268S, Y269F, Q270N, K433N, R488Q and R490Q) within a recently described RNA-binding groove, referred to as the 8x mutant herein (38). Discontinuous cleavage assays were performed in a buffer containing 25 mM bis-Tris propane (pH 8.3), 100 mM NaCl, 15 mM MgCl 2 , 0.1% (v/v) Triton X-100, 1 mM dithiothreitol (DTT) and 32 U of RNaseOUT™ ribonuclease inhibitor (Invitrogen). Reactions were started by combining enzyme (in reaction buffer) with RNA substrate, both of which had been pre-incubated separately at 37 • C for 20 min. Aliquots were taken at each time point and quenched by adding to an equal volume of 2× RNA loading dye: 95% (v/v) formamide, 0.025% (w/v) bromophenol blue, 0.025% (w/v) xylene cyanol and 0.025% (w/v) sodium dodecylsulphate (SDS). The samples were analysed by denaturing PAGE and stained using ethidium bromide.

Kinetic analyses
For the Michaelis-Menten analysis, reactions were performed under similar conditions to those above. Final substrate concentrations were: 0.5, 1, 2, 4, 6, 8 and 10 M. The final enzyme concentration was 40 nM. Samples were separated and analysed by denaturing PAGE alongside a series of dilutions of the substrate at concentrations of 0, 6.25, 12.5, 25, 50, 100 and 500 nM. Initial rates of product formation were calculated from time points in the linear phase of the reaction using the calibration curve of the serial dilutions. These rates were then fitted by non-linear regression to the Michaelis-Menten equation as shown in Equation 1: where v represents the initial rate, [E] represents the total enzyme concentration, k cat represents the enzyme turnover number, [S] represents the initial substrate concentration and K M represents the Michaelis constant.
For the single-turnover experiment, final substrate and enzyme concentrations were 5 nM and 200 nM, respectively. Samples were taken at the following time points: 0, 5, 15, 30, 60 and 120 s, and separated by denaturing PAGE, as described above. Gels were stained with SYBR ® Gold Nucleic Acid Stain (Thermofisher) and visualized on a Fujifilm FLA-5000 scanner with a Y510 filter and the excitation laser set to 478 nm. (v/v) Triton X-100 in a final volume of 240 l. Derivatives of these substrates have been described previously (25,42). The solutions were placed in a quartz cuvette with a path length of 1 mm. The cuvettes were placed into a Jasco J-715 spectropolarimeter and allowed to equilibrate to 37 • C for 10 min. Two circular dichroism (CD) scans were performed at 50 nm/min over a range of 220-320 nm with a 2 s response time, 1 nm pitch and 1 nm bandwidth. The slit width was set to 1000 m. The CD spectrum of the buffer was taken first and was subtracted from the average spectrum of each oligonucleotide. Molar ellipticity was then calculated using the near UV equation as shown in Equation 2: (2) where [θ ] represents the molar ellipticity, θ represents the ellipticity measured, c represents the molarity and l represents the path length of the cuvette. Data were then zerocorrected at 320 nm. High tension values remained below 450 mV for all experiments, indicating a high signal to noise ratio.

Binding assays
Ligand binding assays (LBAs) consisted of a 2-fold dilution series of NTH-RNase E in 25 mM bis-Tris propane (pH 8.3), 100 mM KCl, 15 mM CaCl 2 , 0.1% (v/v) Triton X-100, 1 mM DTT and 20% (v/v) glycerol. Each reaction (final volume of 20 l) contained final reporter concentrations of 20 nM for transcripts for LU13 or 7.5 nM for BR15. Reactions were incubated at 37 • C for 20 min before separating in 1% (w/v) agarose gels in 1× TBE at 10 V/cm for 35 min. LBA gels consisting of transcripts as reporters were stained with SYBR ® Gold Nucleic acid stain. Gels were visualized on the Fujifilm scanner as described above. Band intensities were quantified using ImageJ (43) and used to plot graphs of bound RNA (intensity of bands corresponding to bound species normalized against total band intensities) as a function of NTH-RNase E concentration. K d values were calculated as the concentration of RNase E when 50% of the transcript is bound to the enzyme, as determined by plotting a logistics curve using OriginPro (44).
Competition binding assays (CBAs) consisted of a 2-fold dilution series of the competing transcript prepared in a similar manner to LBAs. Each reaction (final volume of 20 l) contained final concentrations of BR15 (as quadruplex) and NTH-RNase E of 7.5 nM and 20 nM, respectively. The concentration of competitor RNA in the dilution series can be found in each figure legend. Assays were performed and analysed in a similar manner to LBAs. IC 50 values were calculated as the concentration of competing RNA required to cause dissociation of 50% of the BR15 from the NTH.

Cell viability assays
Competent E. coli CJ1832 cells (46) were transformed with the plasmids shown in Table 1. Cultures were plated onto LB agar plates supplemented with 25 g/ml kanamycin and 1 mM isopropyyl-␤-D-thiogalactopyranoside (IPTG), and Empty vector control (45) incubated overnight at 37 • C. To assess colony-forming ability, three separate colonies of each strain were resuspended in 50 l of Luria-Bertani (LB) broth and were streaked on LB agar plates supplemented either with kanamycin only or with kanamycin and IPTG, followed by incubation at 37 • C overnight.
To assess growth in liquid medium, three separate colonies of each strain were resuspended in 500 l of LB broth and 2 l of that was used to inoculate 200 l of LB broth supplemented with either kanamycin only or kanamycin and IPTG. Growth was monitored in a clear 96-well microtitre plate (Greiner Bio-One) by measuring the OD 600 nm of the culture using a FLUOstar Omega plate reader (BMG Labtech) with orbital shaking at 200 rpm between readings.

Multiple-sequence alignment
The amino acid sequences of E. coli K12 RNase E (accession number P21513-1) and RNase G (accession number P0A9J0-1) were obtained from UniProtKB. Amino acid sequences of the closest RNase G homologue from Xylella fastidiosa, Pseudomonas aeruginosa, Haemophilus influenzae, Pasteurella multocida, Vibrio cholera, Yersinia pestis and Salmonella enterica were obtained by BLASTp analysis using the E. coli RNase G sequence. A multiple-sequence alignment of all RNase E/G amino acid sequences was performed using Clustal Omega. Data weres visualized using Jalview (version 2.11.2.2).

Cleavage of tRNA precursors by RNase E is dependent on the presence of an adjacent tRNA unit
Previous analysis of the cleavage of tRNA precursors by RNase E revealed multiple examples where access to a single-stranded region on one side of a tRNA enhanced cleavage on the other, independently of interaction with a 5 -monophosphorylated end (13,14). However, further dissection of three tRNA precursors revealed an additional determinant of RNase E cleavage (Figure 1; see Supplementary Figure S1 for nascent substrates). Analysis of the glyV-glyX-glyY transcript ( Figure 1A) revealed that efficient  Figure S1A). The tRNA units are labelled and coloured. The names of the substrates are indicated at the right, which are coloured black if these substrates were shown to be cleaved by RNase E or grey if they were not cleaved. The numbers that are incorporated into the names of the substrates indicate their size (nt). The sequences of oligonucleotides used to generate the templates for in vitro transcription are provided in Supplementary Table S1. Below this schematic are the cleavage assay results. Reactions were performed as described in the Materials and Methods using the NTH half of RNase E. RNase E cleavage on the 3 side of glyY-tRNA requires, in addition to an accessible single-stranded region on the 5 side of glyY-tRNA (13), the upstream glyX-tRNA. The glyV-tRNA, the first of the tricistronic operon, does not appear to contribute to cleavage on the 3 side of glyY-tRNA, suggesting that a tandem arrangement of tRNA may be sufficient. Analysis of the first half of the tetracistronic transcript argX-hisR-leuT-proM revealed that efficient RNase cleavage on the 3 end of argX-tRNA, which lacks an upstream tRNA as argX-tRNA is the first tRNA, requires access not only to the 5 leader (14) but also to hisR-tRNA, which is immediately downstream ( Figure 1B). This result was shown within the context of a transcript truncated on the 3 side of hisR-tRNA. In a manner similar to the RNase E cleavage 3 to glyY-tRNA, cleavage on the 3 side of metU-tRNA within the heptacistronic transcript metT-leuW-glnU-glnW-metU-glnV-glnX requires not only a singlestranded region on the 5 side of metU-tRNA (13) but also the upstream glnW-tRNA ( Figure 1C). The above experiments indicated that tRNAs adjacent to tRNAs bordered by single-stranded regions that are recognized by RNase E can be important determinants of cleavage efficiency.

Adjacent tRNA units bind to RNase E at a site distant from catalysis
The ability of RNase E to bind tRNA substrates was then investigated using a D346N mutant of NTH-RNase E, which shows a substantial reduction in catalytic activity as a consequence of the substitution of an aspartate required for coordination of the active-site magnesium ion (34). Thus, it was possible to study binding in the absence of detectable substrate cleavage. RNase E was able to bind glnW-tRNA that lacked single-stranded regions on both the 5 and 3 side (Figure 2A). The K d value of 56 nM for this interaction was only 2-fold higher than the K d value of 27 nM for the glnW-metU fragment, which includes single-stranded regions shown to contribute to RNase E cleavage ( Figure  2B). The results suggested that tRNAs as well as flanking single-stranded regions contribute to RNase E binding. Consistent with this suggestion, the K d value of 19 nM for metU-tRNA with flanking single-stranded regions was lower than the K d value of 56 nM obtained for glnW-tRNA without flanking single-stranded regions ( Figure 2B). The smearing toward the top of the gel at the highest concentrations of D346N (compare lanes 16 and C 2 ) is caused by nonstoichiometric amounts of contaminating RNA in NTH-RNase E preparations (estimated to be <0.01% by mass). The affinities of the interactions assayed above are similar to that of RNase E with 5 -monophosphorylated LU13 (K d value of 56 nM, Supplementary Figure S2), a synthetic reference substrate (25,42,47,48) known to be cleaved rapidly by wild-type RNase E via interaction with the 5 -sensor pocket and the channel that binds single-stranded RNA (see the Introduction). In the absence of the 5 -monophosphate group (i.e. when only a single single-stranded region is present), the affinity of RNase E for LU13 decreases by at least 50-fold (K d of >10 M; Supplementary Figure S2). These binding results for LU13 are consistent with previous work that showed that a 5 -monophosphate group does not enhance the turnover number of RNase E and instead decreases the K M (14).
To determine whether binding of glnW-tRNA to RNase E involved regions of the protein also involved in the recognition of single-stranded regions of RNA, we used a competition-binding assay (CBA) with another synthetic reference substrate BR15. This substrate presents four single-stranded regions through the formation of a stable G-quadruplex at the 5 end (25), which was confirmed by CD (Supplementary Figure S3). Prior to being used in the CBA, it was confirmed that the interaction of BR15 and NTH-RNase E has a relatively high affinity (K d of 7.8 nM) and is susceptible to competition by a substrate that presents a single single-stranded region provided the 5 end was monophosphorylated (Supplementary Figure  S4).
The glnW-metU segment and the subfragment that contained metU-tRNA flanked by single-stranded regions, but not glnW-tRNA without flanking single-stranded regions, were able to compete effectively with BR15 for binding (Figure 3). The IC 50 values for the glnW-metU segment and the subfragment that contained metU-tRNA flanked by singlestranded regions were similar (229 nM and 147 nM, respectively), whilst the IC 50 value for glnW-tRNA was at least a magnitude higher (>2.5 M). The higher IC 50 value for glnW-tRNA (compared with the values of the other two substrates) could not be accounted for by the smaller differences in K d values (Figure 2). Considered together, the above results suggested that RNase E binds to glnW-tRNA using a site that does not overlap functionally with the channel that binds single-stranded regions.

Adjacent tRNA units contribute to cleavage by RNase E via an allosteric mechanism and can be substituted by simple stem-loops in mRNA
To explore further the contribution of glnW-tRNA, we undertook a Michaelis-Menten analysis of RNase E cleavage of metU-tRNA flanked at both its 5 and 3 ends by single-stranded regions and the larger glnW-metU fragment, which also includes glnW-tRNA ( Figure 4A). K M values of 3.2 and 3.7 M were obtained for these substrates, respectively, which is consistent with both having similar K d and IC 50 values (Figures 2 and 3, respectively). The corresponding k cat values were 3.35 × 10 −3 /s and 1.85 × 10 −2 /s, respectively. The 5.5-fold difference in these values suggested that the interaction with glnW-tRNA at a site not overlapping with the site of catalysis (as shown in Figure 3) is allosteric and increases catalytic turnover. Moreover, under conditions of enzyme excess, which minimize any effect of differences in product release (49), the rate of cleavage 3 to metU-tRNA was 12-fold faster when glnW was present upstream ( Figure 4B). The cleavage rates in the absence and presence of glnW-tRNA upstream were 1.16 × 10 −5 /s and 1.43 × 10 −4 /s, respectively. This suggests that the presence of glnW-tRNA may reduce the rate of product release and the level of allosteric activation may exceed that indicated by Michaelis-Menten analysis ( Figure 4A).
In order to explore the substrate requirements of tRNAs in binding to and allosterically activating RNase E, modified fragments of tRNA precursors were generated and sub- jected to further cleavage assays ( Figure 5). Removal of the T or D arm or both of these arms, of hisR-tRNA on the argX-hisR fragment did not affect the ability of RNase E to cleave at its site downstream of argX ( Figure 5A), despite the fact that removal of the entire hisR-tRNA does abolish cleavage ( Figure 1B). Given that removal of both arms of the tRNA could potentially form an RNA secondary structure that was more similar to a basic stemloop, a further substrate was generated that replaced the entire hisR-tRNA with the Rho-independent transcriptional terminator of the rpsO transcript. Cleavage of the RNA fragment downstream of the argX-tRNA even when the adjacent hisR-tRNA was replaced with the rpsO terminator was still prominent (Figure 5B), suggesting that the allosteric site on RNase E can interact with relatively simple stem-loops.

The allosteric site is associated with a recently discovered RNA-binding groove, which is essential for cleavage of long RNA substrates and cell viability in E. coli
In parallel with the functional studies presented here, others had identified through X-ray crystallographic studies a groove on RNase E that binds stem-loops within  small regulatory RNAs (38). To determine if this groove binds tRNA, a variant of RNase E (termed the 8x mutant) was generated by introducing eight amino acid substitutions simultaneously along the length of this groove (R3Q, Q22N, H268S, Y269F, Q270N, K433N, R488Q and R490Q), which is non-overlapping with the sites of catalysis and the 5 -sensor, and encompasses the RNase H and small domain ( Figure 6A). These substitutions did not affect the activity of the protein for 5 -monophosphorylated substrates, as observed using a Michaelis-Menten analysis with the substrate 5 -monophosphorylated LU13 ( Figure  6B). K M and k cat values of 6.28 M and 1.93/s, respectively, were obtained for the mutant with the eight substitutions in the groove. These values are similar to those reported for wild-type RNase E (14,25,50). In contrast, mutation of residues within the groove resulted in complete abolishment of cleavage of the argX-hisR-leuT-proM precursor ( Figure  6C). The same groove substitutions also reduced the affinity of RNase E for the rpsO stem-loop ( Figure 6D). Thus, the groove can bind a range of structured RNA segments and is likely to be a site of allosteric regulation of RNase E cleavage.
The finding that the substitutions within the groove had a substantial effect on the cleavage tRNA precursors predicted that they would also affect the growth and possibly the viability of E. coli given that RNase E cleavage activity is essential (24,48,51). This was tested in the context of fulllength RNase E and an established system (45,46) to avoid the need to account for growth effects caused by the trun-cation of RNase E (48,52,53) and to allow direct comparison with previously published work (45,52). Mutations in the RNA-binding groove were introduced into the plasmid pRNE1000, which encodes the full-length RNase E under the control of the constitutive IS10 promoter and without autoregulation (45). To provide a control for reduced cell viability, site-directed mutagenesis was also used to introduce a stop codon after the codon encoding amino acid 529 in a wild-type sequence. The corresponding plasmids were in-  4, 4.9, 9.8, 19.5, 39, 78, 156.3, 312.5, 625, 1250, 2500 and 5000 nM NTH-RNase E, respectively. Lanes C 1 and C 2 contain only 20 nM RNA fragments and only 5000 nM NTH-RNase E, respectively. The top and bottom panel correspond to assays using wild-type or mutant (8×) RNase E, respectively. troduced into the E. coli strain CJ1832 (46), which contains a chromosomal copy of RNase E that has been placed under the control of the lac promoter. Growth was detected on all plates in the presence of IPTG, indicating that there was no dominant-negative phenotype associated with the presence of the mutants. Strains harbouring the wild-type sequence, but not the sequence containing substitutions in the groove, were able to grow in the absence of IPTG (Figure 7A), indicating that the functional groove is essential for growth. As expected, the NTH of RNase E was insufficient to support normal growth within this system. The C-terminal half of RNase E has many important functions including sites of interaction with other proteins that drive assembly of the RNA degradosome complex (52,53). Similar results were found using liquid cultures ( Figure 7B).
The RNA-binding groove may represent a key point of evolutionary divergence of RNase E from its paralogue RNase G, which does share sequence and structural homology but has limited functional overlap (41,(54)(55)(56). Previously it has been shown that many residues of the groove found in E. coli RNase E were conserved in the orthologues of other bacterial species (38). However, an expanded sequence alignment revealed little to no conservation of these residues between E. coli RNase E and RNase G (Supplementary Figure S5). RNase G homologues found in various ␥ -proteobacteria were found to have conserved residues at these positions of a chemical nature less likely to make strong contacts with RNA. The arginine at position 3 in RNase E is conserved as an aspartate in RNase G orthologues, which reverses the charge at this position and could potentially disrupt ionic interactions with the RNA phosphodiester backbone. The glutamine at position 22 in RNase E is conserved as a glycine in RNase G, which would eliminate a potential hydrogen bond between the R group of the glutamine and the RNA. The histidine at position 268 in RNase E is present as leucine, valine or methionine in RNase G, which could all eliminate the ability to form hydrogen bonds with RNA. The tyrosine as position 269 seems to be conserved between RNase E and RNase G. The glutamine as position 270 in RNase E is present as either a glycine or an aspartate in RNase G. The residue lysine at position 433 in RNase E is mostly conserved as a leucine or alanine in RNase G. The arginine at position 488 in RNase E is mostly conserved as a leucine, valine or alanine in RNase G. The arginine at position 490 in RNase E is not present in RNase G, as all the peptide sequences for the orthologues shown here end before this amino acid. The last three residues associated with the RNA-binding groove of RNase E are similarly missing from RNase G and would be predicted to form strong ionic contacts with the phosphodiester backbone of the RNA. In contrast, other residues that are highly conserved in RNase E homologues, such as those involved in 5 -monophosphate interactions (R169 and T170) (34,39,48,57), the active site (F57, F67, D303 and D346) (34,57) and Zn link formation (C404 and C407) (34,58) are also found in RNase G and are conserved between RNase G homologues in other bacteria. Given that RNase G does not appear to be involved in the processing of tRNA or cleavage of many mRNA substrates (13,54,56) and is not essential for cell viability (56,59), these findings suggest that the RNA-binding groove may be a major fea-ture that distinguishes RNase E and RNase G both functionally and phylogenetically.

DISCUSSION
It has been shown here that the binding of tRNAs via a recently discovered groove in the N-terminal catalytic half of E. coli RNase E is a major determinant of the rate of processing of tRNA precursors (Figures 1-7). Moreover, the binding of structured elements is likely to be a widespread determinant of cleavage by E. coli RNase E. The groove that binds structured elements was located through co-crystallization of RNase E with small regulatory RNAs (38) and has been implicated in the generation of the 3 -UTR-derived small RNA MicL (60). Interaction with structured regions, which has long been implicated in the recognition and cleavage of RNA by RNase E (61-64), alongside interaction with single-stranded regions and 5 -monophosphorylated ends (13,14,19,25,36,39,42), may allow RNase E to recognize, cleave and remain associated with substrates via multiple and changing combinations of cooperative interactions in many aspects of RNA biology.
Interaction with the recently discovered groove, which is at distance from the site of catalysis, appears to stimulate RNA cleavage by an allosteric mechanism ( Figures  2-4). It is interesting to note that a kink of ∼40 • within the dimer-dimer interface is one of the major differences in the quaternary structure of the RNase E tetramer in the 'open' apoprotein form (39) compared with a 'closed' holoprotein form in which 5 -monophosphorylated oligonucleotides are bound via the 5 -sensor and the composite single-stranded RNA-binding channel formed by the DNase I and S1 domain (34). Each side of the dimerdimer interface is composed of a pair of 'small' domains linked via chelation of a shared zinc (58). We suggest as a working model that interaction of RNA with the groove, which is a composite of surfaces from the small domain and the RNase H domain in the remainder of the NTH of RNase E, may reduce the kink, thereby promoting the adoption of the 'closed' confirmation, which in turn would enhance cleavage. What is being proposed is essentially a modified 'mouse-trap' model (34) in which conformational closing can be promoted by RNA interacting with the groove.
The recent study of the processing of the 3 -UTR-derived small RNA MicL has found a requirement for two stemloops 3 to the site of cleavage (60). Thus, as found here for tRNA [ Figures 1 and 4; see also (14)], a tandem arrangement of structured elements may be a requirement for efficient RNase E cleavage at many sites including those at a distance from native 5 ends. There is now no need to invoke models in which efficient cleavage within intergenic regions (13,14,25), 3 -UTRs (25)(26)(27)65) and other similarly distant sites (13,66,67) requires RNase E to remain tethered to a 5 -monophosphorylated end (8) with the intervening RNA being looped out (27). It seems more likely that within these regions structured segments in combination with single-stranded regions (13,14,25,26) contributed to 'direct entry' (25,26,32). The finding that RNase E can recognize a transcriptional terminator as part of an interaction Figure 7. Complementation of RNase E deficiency to assess cell viability. CJ1832 strains contained the pRNE1000 plasmid encoding either full-length or NTH RNase E with the wild-type sequence or 8× substitutions. Full-length wild-type is indicated as black, full length 8× mutant as cyan, NTH wild-type as dark green and NTH 8× as red. CJ1832 containing the empty pMPM-k1 vector was included as a control and is shown as dark blue. (A) LB agar plates streaked with three separate colonies of the indicated strains with or without IPTG supplementation (1 mM). (B) Growth curves from liquid culture measured at OD 600 nm with (left) or without (right) IPTG supplementation. Three separate replicates were used for growth curves and the average curve is displayed with the standard error shown as solid lines. Western blots were performed to confirm similar expression levels between all the constructs (data not shown). that produces a cleavage upstream of the terminator (Figures 5 and 6) suggests that RNase E may have a widespread role in initiating 3 to 5 degradation. It is well established that transcriptional terminators impede the progress of 3 to 5 exonucleases (68)(69)(70)(71).
Given the considerable capacity to bind RNA via a large number of combinations of individual sites, the question arises as to why the normal rates of decay of some transcripts are dependent on the RppH RNA pyrophosphohydrolase (22). The answer may simply be that in these but not all cases an interaction with a 5 -monophosphorylated end makes a substantial, non-replaceable contribution to RNase E cleavage. The close coupling of translation and transcription in E. coli and at least some other bacteria (72) means that much of the length of mRNAs will be protected by ribosomes (29,30,32), limiting the contacts initially available to 5 -UTRs. This is not to say that all mRNA degradation initiated at the 5 end may require the generation of a 5monophosphorylated group: RNase E has been mapped in E. coli cells to 5 -UTRs of many mRNAs (30,31,73,74) and binds to a number of 5 -triphosphorylated UTRs in vitro (our unpublished data).
The study of tRNA processing in vivo has revealed that RNase E is not involved in the maturation of all tRNAs (10,11,(75)(76)(77) despite the presence of tandem arrangement of tRNAs or tRNA and transcriptional terminators. This suggest that other features are important determinants of cleavage, probably a combination of the spacing of structure elements, the accessibility of single-stranded regions and 5 -monophosphorylated ends, and the sequence of singlestranded regions accessible by the catalytic site. There are many other cases where individual features that are recognizable by RNase E are present on RNAs but appear to be insufficient to promote cleavage; for example, whilst it appears that many small regulatory RNAs deliver RNase E to specific sites in mRNA (73), there is a class of small RNAs that activate translation without initiating rapid RNA decay (78). A better understanding of the overall control of RNase E cleavage will almost certainly require atomistic descriptions of further RNase E-RNA complexes. Although progress is being make with computational protein-RNA docking, it is hampered by the inherent flexibility of RNA, the transient nature of many protein-RNA interactions and the propensity for global rearrangement upon binding (79).

DATA AVAILABILITY
The data underlying this article are available in the article and in its online supplementary data.